Project Summary Although numerous ribonucleases have been identified over the past 40 years, most have been associated primarily with the biological reaction that was used for their identification. Thus an enzyme like RNase P has been assumed to be strictly involved tRNA processing. Likewise the RNase Z family of enzymes has also been thought to be only involved in tRNA processing, while RNase III type proteins, at least in prokaryotes are considered rRNA maturation enzymes. However, more recent experiments have demonstrated that most of these ribonucleases have multiple functions in the cell. When one carefully looks at what is known about the pathways of rRNA maturation, tRNA processing and mRNA decay, it becomes clear that many of the existing models for these processes are too simplistic and in some cases probably incorrect. In addition, not much is known regarding the enzymatic overlap among these important pathways. For example, during the current grant period, we have demonstrated the existence of multiple new pathways for tRNA processing that require either RNase P or polynucleotide phosphorylase (PNPase) as the first step in processing tRNA precursors rather than RNase E. Furthermore, we hve shown a link between polyadenylation and functional tRNA levels as well as the existence of a previously unidentified endonuclease. Accordingly, this application describes a series of experiments that will focus on developing a more complete understanding of post-transcriptional RNA metabolism in the model prokaryote, Escherichia coli. Our approach will be to use a combination of unique bacterial strains, high density tiling microarrays as well as other molecular biological, biochemical and bioinformatic approaches. Specific experiments include: 1. Transcriptome-wide analysis of the initiation of mRNA processing and decay; 2. Detailed analysis of the role of tRNA nucleotidyl transferase in RNA metabolism and identification of structural features for exonuclease-independent tRNA maturation;3. Characterization of YhgF endonuclease activity and the molecular mechanisms of RNase III- independent 30S rRNA processing. With the increasing prevalence of antibiotic resistant bacteria, the need to better understand the overall mechanism of post-transcriptional RNA metabolism is becoming increasingly important. Information gained from this work could be instrumental in the identification of potential new drug targets.